Project 2 Summary Project 2 addresses transcriptomic signatures of radiation exposure and injury in the following themes: Beyond Simple Exposures: Gene expression signatures will be tested for reconstruction of dose in complex exposure scenarios that mimic those expected to be encountered in an actual radiation / nuclear event, such as a ground-burst detonation of an improvised nuclear device in an urban area. In such an event, exposures will be complicated by the very high dose rate of exposure during the initial flash, decreasing dose rate from fallout, and the presence of neutrons and partial shielding determined in part by the urban architecture. Unique exposure facilities will be used to mimic these realistic scenarios and to test dose reconstruction using a reference transcriptomic signature. Signature genes will be replaced or added to improve both dose reconstruction performance and characterization of complex exposures. Beyond Dose: To address the late effects of radiation, another transcriptomic signature has been developed that can predict death or survival following photon-induced pneumonitis. The impact of mixed neutron+photon exposures on such lung injury is not well known, however, and will now be characterized, with the outcome-predictive transcriptomic signature being tested for mixed neutron+photon exposures. The contribution of senescent cells to the development of pneumonitis and the expression of outcome predictive genes after photon or neutron+photon exposures will also be evaluated. Beyond Model Systems: Biodosimetry is ultimately intended for use with in-vivo human exposures, but assay development typically uses either ex-vivo irradiated human blood or in-vivo irradiated mice or non- human primates. A major gap in knowledge thus exists, regarding the application of results from experimental models to humans. Direct comparison of ex-vivo and in-vivo exposures to both photons and neutrons will be conducted in both non-human primate and mouse models, and the accuracy of dose reconstruction using transcriptomic signatures will be assessed. These studies will include juvenile, adult, and old mice, to quantify the possible impact of age on dose reconstruction, and to test if the ex-vivo model reflects age-specific differences seen in vivo. Non-human primate to human extrapolation will also be addressed, comparing results from ex-vivo neutron irradiated non-human primate and human blood samples to develop and test cross- species conversion approaches for dose reconstruction after neutron exposure. Optimized Biomarker Integration: Data from the biodosimetry approaches of all three Projects (Project 1: cytogenetics, Project 2: gene expression, Project 3: metabolomics) will be analyzed in conjunction to determine the relative strengths of each approach, and to develop decision trees for guiding the application of biodosimetry methodologies in real world situations.